The goal of this application is to elucidate the molecular basis for retro-translocation of the cholera toxin (CT) A1-chain from within the lumen of the endoplasmic reticulum (ER) to the cytosol. CT produced by Vibrio cholerae breeches the intestinal epithelial barrier and enters host epithelial cells to cause disease. CT belongs to the AB family of toxins, where the A component is enzymatically active and the B component is responsible for binding the membrane and mediating entry of the toxin into the cell;traveling from the plasma membrane to the Golgi and ER. After arrival in the ER, a portion of the A-subunit is unfolded and dissociated from the B-subunit by the ER chaperone PDI, targeted to a protein conducting channel, and transported to the cytosol. Shiga toxin and ricin follow a similar pathway. Each of these toxins has evolved structurally to exploit the normal quality control function of the ER that identifies and degrades terminally misfolded proteins in the biosynthetic pathway;the process is termed ERAD for ER associated degradation [3-5]. This process, on which my grant focuses, is termed retro-translocation. In Aim 1, we will use an N-terminally extended mutant toxin reversibly deficient in retro-translocation to explain how the unfolded A1-chain moves from the ER to the cytosol. The failure of this mutant to enter the cytosol suggests that the N-terminal domain is required for the retro-translocation reaction. We will use in vitro systems to define the structure-function relationships of this domain, and to identify other proteins that might interact with the N-terminus of CTA. Reverse genetics using siRNA technology will be used to test dependence on certain proteins known to be required for retro-translocation in other experimental systems. In Aim 2, we will establish an in vitro biochemical assay for retro-translocation of the CT A1-chain using ER proteoliposomes and a mutant CT allowing for assay of retro-translocation by protease-protection. Such an assay would allow complete control over retro-translocation conditions so that individual reactions and necessary and sufficient components could be identified and defined with clarity. The assay would also allow for both targeted and non-biased discovery experiments, and will have high impact in the field. The significance of these studies pertains to their relevance to epithelial mucosal biology and a broad range of clinically important diseases, including acute infectious diarrheas and inflammatory bowel disease. Specifically, CT is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera, which remains prevalent in many parts of the world with poor sanitary conditions and inadequate clean water. Cholera outbreaks can also occur in areas affected by natural disasters, wars and famines [1].